tbuckley/cabot-search huggingface dataset:
Abstract P1072: Hypoxia Inhibits Cellular Contractility Via Allosteric Regulation Of Microtubule Acetylation
Authors: Abhijit Deb Roy, Evan G Gross, Gayatri Pillai, Shailaja Seetharaman, Sandrine Etienne-Manneville, Takanari Inoue
Journal: Circulation Research (2022)

Hypoxia is implicated in pathogenesis of many cardiovascular diseases (CVDs), including loss of cardiac contractility; but its effects on cytoskeletal dynamics are not well understood. The microtubule (MT) network provides mechanical strength to cardiomyocytes and its dysregulation is observed in many CVDs. Acetylated MTs provide structural flexibility to cardiomyocytes and protect against proteinopathy-induced cardiac failure. MT acetylation is exclusively catalyzed by α-TAT1, but little is known about how α-TAT1 is regulated. Here we report that hypoxia inhibits MT acetylation and promotes nuclear accumulation of α-TAT1. We show that cytosolic localization of α-TAT1, disrupted in hypoxia, is critical for MT acetylation. Using computational, live cell microscopy and biochemical approaches, we identified a conserved localization motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements - nuclear export, nuclear import, and cytosolic retention. Inhibiting Exportin 1 induced nuclear accumulation of α-TAT1. We also found that α-TAT1 cytosolic localization is mediated by CDK1, CK2 and PKA kinases, and inhibited by PP2A phosphatase, through a critical residue: T 322 that binds to 14-3-3 proteins. α-TAT1 knock-out (KO) fibroblasts show defects in actomyosin contractility and focal adhesions. We developed an optogenetic tool, named optoATAT1, which rapidly and reversibly shuttled from nucleus to cytosol on blue light stimulation. optoATAT1 stimulation in HeLa cells increased MT acetylation unlike those kept in dark, validating the tool. optoATAT1 stimulation also led to increase in cell contractility indicated by myosin accumulation and focal adhesion maturation, thus confirming a causal relationship between MT acetylation and cell contractility. Also, α-TAT1 KO fibroblasts are highly proliferative, which may pertain to cardiac hypertrophy. In summary, we have identified a novel role of α-TAT1 C-terminus in regulating MT acetylation and cell contractility under hypoxia. Along with this novel regulatory mechanism of MT acetylation, multiple pharmacological agents identified in this study to modulate spatial regulation of α-TAT1 may offer new insights into the treatment of CVDs.
Abstract P3109: Identification Of Drug Transporter Genomic Variants And Inhibitors That Protect Against Doxorubicin Induced Cardiotoxicity
Authors: Tarek Magdy, Carly J weddle, Davi M Lyra Leite, Hananeh Fonoudi, Paul W Burridge
Journal: Circulation Research (2022)

Multiple pharmacogenomic studies have identified the synonymous genomic variant rs7853758 (G &gt; A, L461L) and the intronic variant rs885004 in SLC28A3 (solute carrier family 28 member 3) as statistically associated with a lower incidence of anthracycline-induced cardiotoxicity. However, the true causal variant(s), the cardioprotective mechanism of this locus, the role of SLC28A3 and other solute carrier (SLC) transporters in anthracycline-induced cardiotoxicity, and the suitability of SLC transporters as targets for cardioprotective drugs has not been investigated. To answer these questions, six well-phenotyped, doxorubicin-treated pediatric patients from the original association study cohort were recruited again, and human-induced pluripotent stem cell-derived cardiomyocytes were generated. Patient-specific doxorubicin-induced cardiotoxicity (DIC) was then characterized using assays of cell viability, activated caspase 3/7, and doxorubicin uptake. We demonstrate that patient-derived cardiomyocytes recapitulate the cardioprotective effect of the SLC28A3 locus and that SLC28A3 expression influences the severity of DIC. The role of SLC28A3 in DIC was then confirmed using overexpression and knockout of SLC28A3 in isogenic human-induced pluripotent stem cell-derived cardiomyocytes. To identify the potential causal variant, Nanopore-based fine-mapping of the SLC28A3 locus was then completed after SLC28A3 resequencing. An extended in silico haplotype and functional analysis followed by base editing, identified a novel cardioprotective single nucleotide polymorphism, rs11140490, in the SLC28A3 locus. Next, using high-throughput drug screening of 1200 drugs in patient-derived cardiomyocytes and whole organism validation in mice, we identify the SLC competitive inhibitor desipramine as protective against DIC. Finally, using eight cancer cell lines, we demonstrate that desipramine does not impede doxorubicin cytotoxicity.
Abstract P3017: Aldehyde Dehydrogenase 2 Augments Adiponectin Signaling In Coronary Angiogenesis In Hfpef Associated With Diabetes
Authors: Bipradas Roy, Guodong Pan, Shailendra Giri, Rajarajan Amirthalingam Thandavarayan, Suresh S Palaniyandi
Journal: Circulation Research (2022)

4-hydroxy-2-nonenal (4HNE), a reactive aldehyde, is elevated in diabetes that decreases coronary angiogenesis and this was rescued by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme that detoxifies 4HNE. Adiponectin (APN), a pleiotropic hormone, has pro-angiogenic properties which was shown to be critical in diabetes and its complications. However, the interaction between ALDH2 and APN signaling in coronary angiogenesis is unknown. Coronary endothelial cell (CEC) damage is the initiating step of diabetes-mediated heart failure with preserved ejection fraction (HFpEF) pathogenesis. Thus, we hypothesize that ALDH2 restores 4HNE-induced downregulation of APN signaling in CECs and subsequent coronary angiogenesis in diabetic HFpEF. First of all, APN significantly increased coronary angiogenesis. However, 4HNE treatment significantly decreased the levels of APN signaling molecules including adiponectin receptor (AdipoR)1, APPL1, phospho-AMPK, AMPK, phospho-AKT, and AKT in mouse CECs. Treatment with disulfiram (DSF), an ALDH2 inhibitor, exacerbated 4HNE-mediated decreases in APN-induced increased coronary angiogenesis and APN-signaling cascades, whereas pretreatment with alda1, an ALDH2 activator, rescued the effect of 4HNE. We employed control (db/m) mice, spontaneous type-2 diabetic (db/db) mice, ALDH2*2 knock-in mutant mice with intrinsic low ALDH2 activity (AL), and diabetic mice with intrinsic low ALDH2 activity (AF) that were created by crossing db/db and AL mice to test our hypothesis in vivo. AF mice exhibit severe HFpEF at 6 months compared to age-matched db/db mice as well as 3 months-old AF mice. At 6m, db/db, AL &amp; AF mice significantly decreased APN response to coronary angiogenesis along with increased serum APN levels and decreased cardiac tissue levels of AdipoR1, APPL1, P-AMPK, and P-AKT implicating occurrence of APN resistance which was higher in AF mice. Alda-1 treatment improved APN-mediated angiogenesis significantly in db/db and AF mice. In conclusion, 4HNE-induces APN resistance and subsequently decreases coronary angiogenesis in diabetic HFpEF which was rescued by ALDH2 activation.
Abstract P2064: High Resolution Single Cell Analysis Of The Developing Human Heart
Authors: Semih Bayraktar, Maria Colzani, Hongorzul Davaapil, Vincent Knight-Schrijver, Patrick Rericha, Jonathan Lee, Xiaoling He, Laure Gambardella, Sanjay Sinha
Journal: Circulation Research (2022)

The human heart is composed of various cell types with distinct embryonic origins and functions. Having an understanding of the components of the developing heart at a molecular level should provide better insights into its function in health and disease. Current single cell studies of human fetal hearts are limited by low cell numbers and poor cell type resolution. Here, we aimed to increase the number of cells studied while adding a spatial resolution. We analysed human hearts aged between post conception weeks of 8 and 11, where apex and base in each were processed, providing spatial resolution on cells constituting the myocardium and the great vessels. Our analysis suggests 6 main cell types in the developing hearts with a total of 28 subtypes and provides a differential expression signature for similar cell types with different origins and anatomical locations, including the smooth muscle cells and endothelial cells of the myocardium and the great vessels. Through immunohistochemistry, we validated our findings on these subtypes, as well as on the cells that were less clearly described before, such sinoatrial node cells or resident macrophages. Cell to cell interaction analysis inferred distinct molecular crosstalk across subtypes, including the cells of the great vessels or the vessels of the myocardium. Our findings suggest a list of transcription factors that might be involved in governing the identity of these cells. Collectively, our analysis provides a better understanding of human heart development, and will inform studies into disease modelling and disease progression.
Abstract P3031: Highly Sensitive Impedance Microsensors Based On Graphene Quantum Dots For The Detection Of Metabolically Active Plaques In Animal Models
Authors: Kaidong Wang, Tzung K Hsiai
Journal: Circulation Research (2022)

Detection of rupture-prone and unstable atherosclerotic lesions is of vital values for patients with acute coronary syndromes or stroke. Despite advances in diagnosis and therapy, sensing of metabolically active but angiographically invisible atherosclerotic lesions remains an unsatisfied clinical challenge but highly desirable. Graphene quantum dots (GQDs) have been widely studied in recent years due to its unique optical and electrical properties, thus providing a huge opportunity for building high-performance sensors. Herein, a highly sensitive impedance microsensor based on platinum nanoparticles (PtNPs) @GQDs was constructed for atherosclerotic lesions assessment, especially for the perception of metabolically active but angiographically invisible atherosclerotic lesions. We designed two-step procedures to prepare PtNPs@GQDs microsensors including electrochemical reduction of GQDs and subsequent electrochemical deposition of ultrafine PtNPs. Atherosclerotic lesions samples at mouse aorta and pig carotid confirm this microsensors with highly sensitive impedance signals and indicated possible atherosclerotic lesions. The equivalent circuits simulation suggested that synergetic PtNPs@GQDs creates more parallel conduction path, thus leading to highly sensitive impedance signals. Our studies offer a universal strategy to prepare high-performance impedance sensors and unique opportunities of translational medicine for early atherosclerotic disease detection and prevention of acute coronary syndromes and stroke.
Abstract P2022: Tipifarnib Mediated Protection By Reduction Of Circulating Exosomes In Pressure Overloaded Cardiac Hypertrophy
Authors: Vandana Mallaredy, Rajika Roy, Zhongjian Cheng, Darukeshwara Joladarashi, Ajit Magadum, Charan Thej Gurrala, Maria Cimini, Grace Huang, Venkata Naga Srikanth Garikipati, Chunlin Wang, May Truongcao, Cindy Benedict, Carolina Gonzalez, Walter J Koch, Raj Kishore
Journal: Circulation Research (2022)

Hypertrophic cardiomyopathy (HCM) in response to pathophysiological stress is one of the leading causes of heart failure. A growing body of evidence emphasizes the crucial role of exosomes and their modulated content in aggravating cardiac damage due to their inherent intercellular cross-talk abilities during cardiac remodeling. However, the role of circulating exosomes in HCM for the trafficking of pathogenic factors and remodeling the cardiac microenvironment is yet unclear. We investigated the effect of systemic exosome inhibition during cardiac dysfunction in a transverse aortic constriction (TAC) model of heart failure using a recently identified exosome biogenesis inhibitor, Tipifarnib initially developed as Farnesyl transferase inhibitor. In this study, 10-week-old C57BL6J male mice were randomized into three groups i.e., Sham, TAC and Tipifarnib treated (10 mg/kg) TAC. The untreated TAC mice gradually developed hypertrophy and had reduced cardiac functions with a significant increase in heart weight/body weight ratio, cardiomyocyte size and upregulation of hypertrophy and fibrosis associated genes expression by 8 weeks. On the contrary, Tipifarnib treated TAC mice showed remarkably improved cardiac left ventricular functions, reduced cardiac hypertrophy and fibrosis. Notably, Nanosight analysis indicated significantly higher serum exosomes concentration in TAC mice which were substantially suppressed with Tipifarnib treatment. The molecular analysis of the heart tissue revealed Tipifarnib treated TAC mice had reduced expression of the proteins involved in exosome biogenesis in comparison to untreated TAC mice. To gain insight into the cargo of these circulating exosomes, we performed the serum cytokine array and serum exosomes miRNA sequencing in untreated and Tipifarnib treated TAC mice. There was a marked reduction in inflammatory cytokines in serum and differentially expressed exosomal miRNAs with Tipifarnib treatment in comparison to the untreated TAC mice. Overall, our studies suggest the promising potential of Tipifarnib that effectively protects against pressure overload-induced cardiac remodeling and dysfunction by suppressing exosome secretion and altering hypertrophic and fibrotic gene expression.
Abstract P3082: Emergency Granulopoiesis Increases Neutrophil Phenotypic And Functional Diversity
Authors: Jonah Stephan, Zhen Gu, Zoubul Taylor, Marcin Wysoczynski
Journal: Circulation Research (2022)

Ischemic injury due to myocardial infarction (MI) elicits immune cell infiltration to remove necrotic tissue and facilitate scar formation. Neutrophils are among the first infiltrating immune cells, and neutrophilia has been commonly accepted as a biomarker of adverse prognoses post-MI. The increased demand for neutrophils is met by an accelerated granulocyte production in a process known as emergency granulopoiesis. However, the impact of emergency granulopoiesis on neutrophil function remains cryptic. These experiments seek to determine the impact of emergency granulopoiesis on neutrophil phenotype and secretory function. To activate emergency granulopoiesis, C57BL/6 mice were injected with G-CSF for 3 d (100 μg/kg/day, s.c.). Neutrophil phenotype and function were evaluated 24 h after the last dose of G-CSF. We found that G-CSF injections resulted in increased neutrophils in peripheral blood, spleen, and bone marrow. Flow cytometric analysis revealed that neutrophils produced via emergency granulopoiesis showed lower expression of the classical neutrophil marker Ly6G and an increased number of CD101 Neg neutrophils in peripheral blood and spleen (n=3-6/group, p&lt;0.05). Functional analysis in vitro showed that neutrophils produced via emergency granulopoiesis are 2-3 times more susceptible to PMA- and fMLF-induced degranulation of primary granule content (neutrophil elastase and myeloperoxidase), but release of secondary (NGAL, CHI3L1), and tertiary granules (MMP9) remained unchanged (n=5/group, p&lt;0.05). Based off these observations, we conclude that emergency granulopoiesis results increased diversity of neutrophil phenotype and function. Future studies will elucidate the impact of emergency granulopoiesis neutrophils on post-MI scar formation and chronic ventricular remodeling.
Abstract P2040: Metolazone As An Adjunct To Loop Diuretics In Acute Heart Failure: A Systematic Review And Meta-analysis
Authors: Dhan B Shrestha, Suman Gaire, Barun B Aryal, Wasey ali Yadullahi Mir, Yub Raj Sedhai
Journal: Circulation Research (2022)

Introduction: Acute heart failure (AHF) affects a significant proportion of the US population. The first-line therapy for decompensated AHF is loop diuretics. However, diuretic resistance in AHF cases is common. In such cases, as add-on therapy, thiazide diuretics are often used to combine two different mechanisms of action. However, the certainty of effectiveness and safety profiles of combining diuretics in cases of diuretic-resistant AHF has not been evaluated in systematic review and meta-analysis considering metolazone, so this study was proposed. Methods: The review protocol was registered in PROSPERO (CRD42022302399). PRISMA guideline was followed. Databases were searched using appropriate keywords for eligible papers published before January 5, 2022. The database was screened using the Covidence platform, and analysis was done using Review Manager (RevMan-5.1) software. Odds ratio or mean difference with 95% CI were estimated using fixed or random effect models based on heterogeneity. Results: Eight studies among 2999 studies met the inclusion criteria (2 RCTs, six observational studies). The pooled analysis showed no difference in standardized mean difference among the metolazone group and the control group for 24-hours of total urine output (SMD 0.10, CI -0.40 to 0.60) and change in 24-hour 48-hour urine output and 72-hour urine output. There was no difference comparing the addition of metolazone or chlorothiazide to furosemide. However, pooling of the result among those studies comparing furosemide and metolazone with furosemide alone showed a significant increase in the 24-hour total urine output metolazone group (SMD 0.50, CI -0.00 to 0.99). Other outcomes like weight loss, mortality, readmission rate, adverse events did not differ across treatment and control groups. Conclusion: Though some benefit in 24-hour urine output was noted on the addition of metolazone to furosemide, our analysis could not demonstrate the benefit of adding metolazone due to lack of adequate studies comparing similar groups in diuretic resistant AHF. Therefore, further RCTs are required in this subject to evaluate the benefit of adding metolazone in diuretic-resistant AHF patients.
Abstract P1092: Voltage Gated Sodium Channel Subunit Mimetic Peptide βadp1 Increases Regulated Intramembrane Proteolysis Of Scn1b/β1
Authors: Zachary J Williams, Daniel Hoagland, Jane Jourdan, Robert G Gourdie
Journal: Circulation Research (2022)

Clinical treatment for cardiac arrhythmias based on targeting ion channels is limited and can have deadly side effects. We previously reported that acute administration of βadp1 resulted in decreased action potential conduction velocity and increased arrhythmias in isolated guinea pig hearts. These changes were associated with disruption of trans-homophilic adhesion of the β1 subunit within gap junction-adjacent perinexal domains in intercalated discs. Using Electric Cell-Substrate Impedance Sensing (ECIS), we found that βadp1 shows opposing acute and long-term effects on β1 mediated adhesion. βadp1 decreases β1 mediated adhesion in 1610 cells stably expressing SCN1B/β1 (1610β1) over the first 24 hours, but after 48 hours is associated with increased adhesion. Regulated Intramembrane Proteolysis (RIP) of β1 sequentially by BACE-1 and γ-secretase results in a cleaved intracellular fragment that translocates to the nucleus and alters transcription of various genes, including VGSC subunits. Therefore, we hypothesized the long-term gain-of-adhesion-function with βadp1 treatment resulted from increased RIP of β1, with downstream VGSC gene expression effects. Inhibition of γ-secretase with DAPT accumulates a C-terminal fragment (CTF) of β1 during RIP, enabling quantification of changes to RIP. Thus, 1610β1 cells were treated with 50μM βadp1, 1μM DAPT, 50μM βadp1+1μM DAPT or 0.1% DMSO control. Cells were fixed for β1 immunofluorescence (IF) or lysate was collected at 6-, 24-, and 48-hours post treatments for Western blotting. DAPT treatment significantly increased CTF levels compared to control at all timepoints (p&lt;.0001). However, βadp1+DAPT treatment resulted in greater accumulation of β1 CTF levels over DAPT alone at each timepoint. (n=6, 6 hr: p&lt;.01, 24 hr: p&lt;.01, 48 hr: p&lt;.05). βadp1+DAPT treatment caused a significant increase in full-length β1 at 6 hours, but no difference at 24 or 48 hours. The data suggest depletion of β1 by increased RIP of β1 after βadp1 treatment. IF showed an increase in abundance of β1 48 hours after βadp1 treatment compared to DMSO. In sum, our data indicates that βadp1 may upregulate RIP of β1 with potential downstream effects on gene transcription of VGSC subunits, suggesting a path to a novel class of antiarrhythmics.
Abstract P2005: Loss Of Full-length Mylk3 Causes Dilated Cardiomyopathy Via A MYL2-independent Mechanism
Authors: Jack L Williams, Anju Paudyal, Michelle Stewart, Pedro Cutillas, Andrew Tinker, Lou Metherell
Journal: Circulation Research (2022)

Introduction: We recently discovered C57BL/6N mice develop dilated cardiomyopathy (DCM), while the closely related C57BL/6J do not. We identified a variant (Chr8:85365179A&gt;T, c.-5T&gt;A) in myosin light chain kinase 3 ( Mylk3 ) in the C57BL/6N substrain that abolishes translation of the predominant transcript (ENSMUST00000034133). MYLK3 phosphorylates many sarcomere proteins, in particular myosin light chain 2 (MYL2), vital for cardiac development and function. Hypothesis: The MYLK3 variant is sufficient to drive the development of DCM. Results: We generated a novel isogenic mouse line carrying the Mylk3 c.-5T&gt;A variant on the C57BL/6J background (C57BL/6J Mylk3.6N ). We found symptoms of DCM in male mutant mice at just 4 weeks old, with increased left ventricular diameter and volume, thinning of the posterior wall and a reduced ejection fraction. These data suggest the Mylk3 variant alone is sufficient to cause DCM. Surprisingly, we found no difference in phosphorylation of myosin light chain 2 (MYL2) by Phos-Tag® blotting at any age. This was unexpected, as previous reports in Mylk3 -/- mice found complete loss of MYL2 phosphorylation with a similar profile of DCM. However, in our mouse model, loss of MYLK3 is caused by a single point mutation in exon 1 which ablates the primary isoform (ENSMUST00000034133; Q3UIZ8) but western blotting with a C-terminal antibody revealed expression of a shorter isoform that is unaffected by the variant due to a distinct N-terminus (ENSMUST00000122452; D3Z630). There was no difference between the isoforms in their ability to phosphorylate MYL2 suggesting the shorter isoform can compensate. MYLK3 may also interact with ACTN2 and phosphorylate TNNI3. We found no difference in the expression or localisation of ACTN2 in our tissues, although co-IP in cells showed the primary isoform binds more readily to ACTN2, however, there was a significant reduction in TNNI3 S23/24 phosphorylation at all ages in C57BL/6J Mylk3.6N mice. This could affect cross-bridge cycling kinetics by altering the function of TNNI3 within the tropomyosin/troponin complex. Conclusions: Loss of full-length MYLK3 causes DCM via a MYL2-independent mechanism, possibly due to lack of phosphorylation of alternative target(s), such as TNNI3.